A fast, efficient chromatin immunoprecipitation method for studying protein-DNA binding in Arabidopsis mesophyll protoplasts

Lee, Jeong‐Hwan; Jin, Suhyun; Kim, Sun Young; Kim, Wanhui; Ahn, Ji Hoon

doi:10.1186/s13007-017-0192-4

Cited by 57 publications

(42 citation statements)

References 58 publications

(68 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We first performed qRT-PCR on total RNA isolated from protoplasts of both NT and OsNAC14 OX plants (Figure 6A ). Similar with stable transgenic plants, all five genes were up-regulated in protoplasts of OsNAC14 OX plants suggesting that the isolated protoplasts maintained transcriptional network of intact plants similar to what was observed by Lee J. H. et al ( 2017 ) (Figure 6A ). Next, we introduced 35S::OsNAC14-MYC or 35S::GFP-MYC effector plasmids into protoplasts isolated from NT plants to verify that the transient expression of OsNAC14 can induce similar response that was shown in stable OsNAC14 OX plants.…”

Section: Resultssupporting

confidence: 82%

“…To confirm whether OsRAD51A1 is a direct target of OsNAC14, we performed chromatin immunoprecipitation (ChIP) coupled with qRT-PCR analysis on rice protoplast system. Protoplasts provide a good platform to perform functional characterization of genes and isolation of protein-DNA complex in plants (Zhang Y. et al, 2011 ; Lee J. H. et al, 2017 ). We first performed qRT-PCR on total RNA isolated from protoplasts of both NT and OsNAC14 OX plants (Figure 6A ).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Overexpression of OsNAC14 Improves Drought Tolerance in Rice

et al. 2018

View full text Add to dashboard Cite

Plants have evolved to have sophisticated adaptation mechanisms to cope with drought stress by reprograming transcriptional networks through drought responsive transcription factors. NAM, ATAF1-2, and CUC2 (NAC) transcription factors are known to be associated with various developmental processes and stress tolerance. In this study, we functionally characterized the rice drought responsive transcription factor OsNAC14. OsNAC14 was predominantly expressed at meiosis stage but is induced by drought, high salinity, ABA, and low temperature in leaves. Overexpression of OsNAC14 resulted in drought tolerance at the vegetative stage of growth. Field drought tests demonstrated that OsNAC14 overexpressing transgenic rice lines exhibited higher number of panicle and filling rate compared to non-transgenic plants under drought conditions. RNA-sequencing analysis revealed that OsNAC14 overexpression elevated the expression of genes for stress response, DNA damage repair, defense related, and strigolactone biosynthesis. In addition, chromatin immunoprecipitation analysis confirmed the direct interaction of OsNAC14 with the promoter of OsRAD51A1, a key component in homologous recombination in DNA repair system. Collectively, these results indicate that OsNAC14 mediates drought tolerance by recruiting factors involved in DNA damage repair and defense response resulting in improved tolerance to drought.

show abstract

Section: Resultssupporting

confidence: 82%

Section: Resultsmentioning

confidence: 99%

Overexpression of OsNAC14 Improves Drought Tolerance in Rice

et al. 2018

View full text Add to dashboard Cite

show abstract

“…The transfected protoplasts were fixed using 1% formaldehyde, quenched with 134 mM glycine, and kept frozen at −80°C. Three replicates were processed for ChIP analysis performed based on Lee et al (2017) and Lau and Bergmann (2015). In brief, nuclei were isolated from the frozen protoplasts and chromatin was fragmented ranging from 100 to 600 bp using a Covaris ME220 Focused ultrasonicator.…”

Section: Chip Sequencing Analysismentioning

confidence: 99%

Response to Persistent ER Stress in Plants: A Multiphasic Process That Transitions Cells from Prosurvival Activities to Cell Death

Srivastava

Russo³

et al. 2018

Plant Cell

View full text Add to dashboard Cite

The unfolded protein response (UPR) is a highly conserved response that protects plants from adverse environmental conditions. The UPR is elicited by endoplasmic reticulum (ER) stress, in which unfolded and misfolded proteins accumulate within the ER. Here, we induced the UPR in maize () seedlings to characterize the molecular events that occur over time during persistent ER stress. We found that a multiphasic program of gene expression was interwoven among other cellular events, including the induction of autophagy. One of the earliest phases involved the degradation by regulated IRE1-dependent RNA degradation (RIDD) of RNA transcripts derived from a family of peroxidase genes. RIDD resulted from the activation of the promiscuous ribonuclease activity of ZmIRE1 that attacks the mRNAs of secreted proteins. This was followed by an upsurge in expression of the canonical UPR genes indirectly driven by ZmIRE1 due to its splicing of mRNA to make an active transcription factor that directly upregulates many of the UPR genes. At the peak of UPR gene expression, a global wave of RNA processing led to the production of many aberrant UPR gene transcripts, likely tempering the ER stress response. During later stages of ER stress, ZmIRE1's activity declined, as did the expression of survival modulating genes, and , amid a rising tide of cell death. Thus, in response to persistent ER stress, maize seedlings embark on a course of gene expression and cellular events progressing from adaptive responses to cell death.

show abstract

“…Primers flanking the ORA59-binding sequence of the AtACT promoter for qRT-PCR are present in Table S1. ChIP assays were strictly performed as recently described (Lee et al, 2017;Zhou et al, 2017).…”

Section: Chip-qpcr Assaymentioning

confidence: 99%

Jasmonic acid/ethylene signaling coordinates hydroxycinnamic acid amides biosynthesis through ORA59 transcription factor

Zhang

Meng

et al. 2018

The Plant Journal

View full text Add to dashboard Cite

Hydroxycinnamic acid amides (HCAAs) are a class of antimicrobial metabolites involved in plant defense against necrotrophic pathogens, including Alternaria brassicicola and Botrytis cinerea. The agmatine coumaryl transferase (AtACT) is the key enzyme that catalyzes the last reaction in the biosynthesis of HCAAs, including p-coumaroylagmatine (CouAgm) and feruloylagmatine in Arabidopsis thaliana. However, the regulatory mechanism of AtACT gene expression is currently unknown. Yeast one-hybrid screening using the AtACT promoter as bait isolated the key positive regulator ORA59 that is involved in jasmonic acid/ethylene (JA/ET)-mediated plant defense responses. AtACT gene expression and HCAAs biosynthesis were synergistically induced by a combination of JA and ET. In the AtACT promoter, two GCC-boxes function equivalently for trans-activation by ORA59 in Arabidopsis protoplasts, and mutation of either GCC-box abolished AtACT mRNA accumulation in transgenic plants. Site-directed mutation analysis demonstrated that the specific Leu residue at position 228 of the ORA59 EDLL motif mainly contributed to its transcriptional activity on AtACT gene expression. Importantly, MEDIATOR25 (MED25) and ORA59 homodimer are also required for ORA59-dependent activation of the AtACT gene. These results suggest that ORA59 and two functionally equivalent GCC-boxes form the regulatory module together with MED25 that enables AtACT gene expression and HCAAs biosynthesis to respond to simultaneous activation of the JA/ET signaling pathways.

show abstract

A fast, efficient chromatin immunoprecipitation method for studying protein-DNA binding in Arabidopsis mesophyll protoplasts

Cited by 57 publications

References 58 publications

Overexpression of OsNAC14 Improves Drought Tolerance in Rice

Overexpression of OsNAC14 Improves Drought Tolerance in Rice

Response to Persistent ER Stress in Plants: A Multiphasic Process That Transitions Cells from Prosurvival Activities to Cell Death

Jasmonic acid/ethylene signaling coordinates hydroxycinnamic acid amides biosynthesis through ORA59 transcription factor

Contact Info

Product

Resources

About